Spermatogenesis is a complex process of differentiation that is clearly dependent upon paracrine interactions among the cellular elements in the testis. The different cells (myoid, germ, Leydig, Sertoli) certainly do not function in isolation, but there is a coordinated combination of events that collectively permit spermatogenesis. Our long term objective is to achieve a better understanding of the factors controlling germ cell differentiation. Our research will emphasize the role of the Sertoli cell in the spermatogenic process and the interdependence of the cellular elements within the testis. During the previous granting period, we developed a novel dual compartment (bicameral) culture system which mimics more closely cells growing in vivo. In this renewal application, we will continue our experiments to characterize the Sertoli cells in bicameral culture and, in particular, attempt to prove that Sertoli cells growing in this culture chamber are more physiological than cells in conventional culture. The effects of other testicular cells on Sertoli cell differentiation will be determined. We have recently demonstrated that the Sertoli cells produce a mitogenic protein which modulates Leydig cell division. We will attempt to determine whether this factor plays a direct role in germ cell mitoses and differentiation. Our recent work demonstrated that pachytene spermatocytes produce a factor(s) which stimulates novel protein secretion by Sertoli cells. We intend to continue our studies on the reciprocal relationship between germ cells and Sertoli cells. The bicameral cell culture chamber has lcd to many novel experiments in the testis, including the ability to recombine simultaneously the cellular elements of the testis in culture in a manner which is analogous to their position in vivo. Sertoli cells will be grown on top of extracellular matrix-coated Millipore filters; myoid cells will be placed on the undersurface of the filters; Leydig cells will be cultured in the basal chamber; different classes of germ cells will be placed on top of the filter with the Sertoli cells. With these experiments we may be able to achieve a greater degree of germ cell differentiation in culture than has been previously possible, especially because of the newly developed bicameral culture chamber. The experiments outlined in this proposal should provide new data on the basic biology of the Sertoli cell and its role in spermatogenesis and on the interdependence of the cellular elements in the testis. This approach could well have significance in the development of future male contraceptives.